Rationale: The functional connectivity analysis reported in the main body of this paper averaged across left and right homotopic portions of each striatal seed region. This methodological choice was based on the following reasons. First, 40/41 patients had bilateral symptoms (i.e. UPDRS score > 0 for both sides and H&Y stage > 2), indicating significant striatal dopamine depletion in both hemispheres. Second, intrinsic fluctuations have been shown to be strongly correlated across bilateral homologues regions (Salvador et al., 2005) and most resting state network are bilateral (Damoiseaux et al., 2006). In particular, intrinsic cortico-striatal fluctuations in healthy humans were found to span both hemispheres, regardless of the hemisphere seeded
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S u p p l. Fig 4 Frequency characteristics o f th e seed regions
The average power spectrum (± SEM) for each of the four seed regions is shown separately for the healthy controls (blue lines) and for the PD patients (pink lines). Analyses were done on unfiltered time courses, after having removed variance attributed to nuisance and tremor regressors using multiple linear regression. The results show that there are no significant differences in the frequency distribution across groups.
(Di Martino et al., 2008). Third, averaging over homotopic regions reduced the impact of outliers on the calculation of the average tim e courses.
Nevertheless, it appears relevant to investigate whether the least- and most-affected basal ganglia differ in terms of their connectivity profile. However, although PD is an inherently asymmetric disease, the asymmetry of the pathology varies between patients and our subjects were not selected on the basis of this characteristic. Therefore, to increase the sensitivity of this particular control analysis, we considered a sub-sample of the patients that had markedly asymmetric symptoms, as defined by an asymmetry index (AI) of >0.2, based on clinical disease severity
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(lateralized UPDRS-III scores). There were 28 PD patients with these characteristics (15 men; mean age of 55.1 + 9.9 years; 18 right- and 10 left-side affected patients; mean lateralized UPDRS-III score: most-affected side, 14.0 ± 0.6 points; least-affected side, 5.4 ± 0.6 points, p<0.001). They were compared to the group of 36 healthy controls. M ethods: The fMRI analysis was the same as described for the bilateral seed regions, except that the first-level analysis included six instead of three striatal seed regressors (i.e. left and right caudate nucleus, left and right posterior putamen, left and right anterior putamen). Statistical comparisons between patients and controls were performed at the second level (random effects analysis), using a statistical model with factors GROUP (PD, controls), SIDE (least-affected, most-affected) and REGION (posterior putamen, anterior putamen, caudate nucleus). The PCC was not included in this analysis, because its midline location does not enable reliable comparisons across hemispheres. In order to average over left- and right-side affected patients, we flipped the contrast images of the 10 left-side affected PD patients in the axial plane. The same procedure was performed in the same proportion (14/36) of healthy controls. Thus, in the PD group, the least-affected hemisphere referred to the left hemisphere in 18/28 patients, while in the control group the "least-affected" hemisphere referred to the left hemisphere in 22/36 subjects. This allowed us to directly compare the two sides across groups, while controlling for the effect of laterality (left or right side).
We focused our analyses on finding GROUP x SIDE interactions for each of the three striatal seed regions, while restricting our search to the brain regions showing a significant GROUP effect in our main analysis (region of interest analysis within two spatial maps: 1) "controls > patients" for the posterior putamen, shown in cyan in Fig. 3 A-C; 2) "patients > controls" for the anterior putamen, shown in yellow in Fig. 3 A-C). This approach increased our sensitivity to find hemispheric differences, and it is justified by the fact that the GROUP x SIDE interaction is orthogonal to the main effect of GROUP.
Results: There were no significant differences for the posterior putamen or the caudate nucleus (no GROUP x SIDE interaction). However, the connectivity profile of the least-affected anterior putamen was specifically increased in the PD group as compared to controls, and this effect was visible in the right inferior parietal cortex (significant GROUP x SIDE interaction; MNI coordinates [46 -28 28], t=4.34, p=0.009 FDR-corrected; Suppl. Fig. 5).
It may seem surprising that there were no laterality differences for the posterior putamen. On the other hand, given that this region is most affected by dopaminergic denervation, which is estimated to be around 80% at the tim e of diagnosis, a flooring effect might explain the lack of differences between least- and most-affected sides (Pirker, 2003). In other words, in the posterior putamen of both hemispheres, dopamine
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S u p p l. Fig 5 cortico-striatal con nectivity of th e least- vs. th e m o st-affected hem isphere
Panel A shows the spatial distribution of the contrast: "patients, un-affected>affected" > "controls, un-affected>affected" for the anterior putamen, thresholded at p<0.001 uncorrected (for graphical purposes) and overlaid on a coronal slice from a representative anatomical image of the MNI series. The red circle marks the right inferior parietal cortex (IPC). Panel B shows the beta values from the right IPC (MNI coordinates [46 -28 28]). Functional connectivity (beta value, on the y-axis) between this region and the posterior and anterior putamen in the least- and most-affected hemispheres (on the x-axis) is shown separately for healthy controls (white bars) and for PD patients (black bars). The results show that the increased functional connectivity with the anterior putamen in the PD group was specific for the least-affected hemisphere.
levels were most likely already below a critical level necessary for normal cortico- striatal connectivity. Interestingly, we found that the increased connectivity profile of the anterior putamen was largest for the least-affected side. This is in agreement with the hypothesis that it reflects a compensatory mechanism: given that dopamine levels are highest in the least-affected anterior putamen, this structure seems most capable of compensating for more dopamine-depleted portions of the striatum.
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